U.S. patent application number 10/749538 was filed with the patent office on 2004-12-09 for remedies for myeloma to be used together with nitrogen mustard antitumor agents.
Invention is credited to Akamatsu, Kenichi, Nakamura, Akito.
Application Number | 20040247621 10/749538 |
Document ID | / |
Family ID | 15847661 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040247621 |
Kind Code |
A1 |
Nakamura, Akito ; et
al. |
December 9, 2004 |
Remedies for myeloma to be used together with nitrogen mustard
antitumor agents
Abstract
A therapeutic agent for myeloma comprising a combined use of a
nitrogen mustard anticancer agent and anti-IL-6 receptor antibody.
Thus, a therapeutic agent for myeloma comprising anti-IL-6 receptor
antibody for use in combination with a nitrogen mustard anticancer
agent; a therapeutic agent for myeloma comprising a nitrogen
mustard anticancer agent for use in combination with anti-IL-6
receptor antibody; and a therapeutic agent for myeloma comprising a
nitrogen mustard anticancer agent and anti-IL-6 receptor
antibody.
Inventors: |
Nakamura, Akito; (Shizuoka,
JP) ; Akamatsu, Kenichi; (Shizuoka, JP) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
15847661 |
Appl. No.: |
10/749538 |
Filed: |
December 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10749538 |
Dec 30, 2003 |
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09202802 |
Dec 22, 1998 |
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6692742 |
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09202802 |
Dec 22, 1998 |
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PCT/JP97/02246 |
Jun 27, 1997 |
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Current U.S.
Class: |
424/200.1 ;
435/252.3 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 2300/00 20130101; C07K 2317/24 20130101; C07K 16/2866
20130101; A61K 38/00 20130101; A61P 35/00 20180101; A61K 39/39541
20130101; A61K 39/39541 20130101; C07K 2319/00 20130101; A61K
39/395 20130101; A61K 39/395 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/200.1 ;
435/252.3 |
International
Class: |
A61K 039/02; C12N
001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 1996 |
JP |
8-167325 |
Claims
What is claimed is:
1. A method for treating myeloma, comprising administering a
nitrogen mustard anticancer agent in combination with an anti-IL-6
receptor antibody as part of a treatment regimen, wherein the
nitrogen mustard anticancer agent is administered in an amount to
enhance the therapeutic effect of the anti-IL-6 receptor
antibody.
2. The method according to claim 1, wherein the anti-IL-6 receptor
antibody is a monoclonal antibody.
3. The method according to claim 2, wherein the monoclonal antibody
is a PM-1 antibody.
4. The method according to claim 3, wherein the PM-1 antibody is a
reshaped human PM-1 antibody.
5. The method according to claim 1, wherein the nitrogen mustard
anticancer agent is mechlorethamine, nitrogen mustard N-oxide,
melphalan, uramustin, ifosfamide, chlorambucil, or
cyclophosphamide.
6. The method according to claim 1, wherein the nitrogen mustard
anticancer agent is melphalan and the anti-IL-6 receptor antibody
and nitrogen mustard, anticancer agent provide a synergistic
effect.
7. A method for treating myeloma, comprising administering an
anti-IL-6 receptor antibody in combination with a nitrogen mustard
anticancer agent as part of a treatment regimen, wherein the
anti-IL-6 receptor antibody is administered in an amount to enhance
the therapeutic effect of the nitrogen mustard anticancer
agent.
8. The method according to claim 7, wherein the anti-IL-6 receptor
antibody is a monoclonal antibody.
9. The method according to claim 8, wherein the monoclonal antibody
is a PM-1 antibody.
10. The method according to claim 9, wherein the PM-1 antibody is a
reshaped human PM-1 antibody.
11. The method according to claim 7, wherein the nitrogen mustard
anticancer agent is mechlorethamine, nitrogen mustard N-oxide,
melphalan, uramustin, ifosfamide, chlorambucil, or
cyclophosphamide.
12. The method according to claim 7, wherein the nitrogen mustard
anticancer agent is melphalan and the anti-IL-6 receptor antibody
and nitrogen mustard anticancer agent provide a synergistic effect.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pharmaceutical
composition for combined use of nitrogen mustard anticancer agents
with anti-IL-6 receptor antibody for treatment of myeloma.
BACKGROUND ART
[0002] For chemotherapy of human tumors, alkylating agents,
antimetabolites, antitumor antibiotics, platinum compounds and the
like have been used. When single uses of these activating agents do
not exhibit marked therapeutic effects, therapies in which multiple
drugs are used in combination have been considered (Frei, E. III,
Cancer Res. (1992) 32, 2593-2607). As anticancer agents that belong
to the alkylating agents, there are mentioned nitrogen mustard
anticancer agents, which is a general term used for the anticancer
agents that have a partial structure called nitrogen mustard. Of
the nitrogen mustard anticancer agents melphalan has been put into
practical use.
[0003] IL-6 is a multifunctional cytokeine called B-cell
stimulatory factor 2 or interferon .beta.2. IL-6 was discovered as
a differentiation factor responsible for activation of
B-lymphocytes (Hirano, T. et al., Nature (1986) 324, 73-76).
Thereafter, it was found to be a multifunctional cytokeine that
influences the function of various cells (Akira, S. et al., Adv. in
Immunology (1993) 54, 1-78). IL-6 imparts its biological activity
through two proteins on the cell membrane.
[0004] One of them is a ligand-biding protein with a molecular
weight of about 80 kD, IL-6 receptor, to which IL-6 binds. IL-6
receptor occurs not only in a membrane-bound form that penetrates
and is expressed on the cell membrane but also as a soluble IL-6
receptor consisting mainly of the extracellular region. The other
is non-ligand-binding gp130 with a molecular weight of about 130 kD
that takes part in signal transmission. IL-6 and IL-6 receptor form
a IL-6/IL-6 receptor complex, to which another membrane protein
gp130 is bound, and thereby the biological activity of IL-6 is
transmitted to the cell (Taga et al., J. Exp. Med. (1987) 166,
967).
[0005] Antibodies to IL-6 receptor (anti-IL-6 receptor antibodies)
have been known (Novick D. et al., Hybridoma (1991) 10, 137-146
Huang, Y. W. et al., Hybridoma (1993) 12, 621-630 International
Patent Application WO95-09873, French Patent Application FR
2694767, U.S. Pat. No. 5,216,128), one of which is PM-1 derived
from mice (Hirata et al., J. Immunology (1989) 143, 2900-2906).
Furthermore, a reshaped antibody obtained by replacing the
complementarity determining regions (CDRs) of the mouse antibody
with the CDRs of a human antibody has also been known.
[0006] However, the combined use of a nitrogen mustard anticancer
agent and IL-6 receptor as a therapeutic agent for treatment of
myeloma has not been known.
DISCLOSURE OF THE INVENTION
[0007] It is an object of the present invention to provide a new
type of myeloma therapeutic agent that is more effective than the
conventionally known myeloma therapeutic agents.
[0008] As a result of an intensive study to solve the above
problems, the applicants have found that the combination of a
nitrogen mustard anticancer agent, a conventionally known
anticancer agent, and anti-IL-6 receptor antibody has a synergistic
effect, i.e. it is more effective than the sole use of the nitrogen
mustard anticancer agent or the sole use of anti-IL-6 receptor
antibody for treatment of myeloma, and have completed the present
invention.
[0009] Thus, the present invention provides a therapeutic agent for
treatment of myeloma comprising anti-IL-6 receptor antibody for use
in combination with a nitrogen mustard anticancer agent.
[0010] The present invention also provides a therapeutic agent for
treatment of myeloma comprising anti-IL-6 receptor monoclonal
antibody for use in combination with a nitrogen mustard anticancer
agent.
[0011] The present invention also provides a therapeutic agent for
treatment of myeloma comprising PM-1 antibody for use in
combination with a nitrogen mustard anticancer agent.
[0012] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a reshaped PM-1 antibody for use in
combination with a nitrogen mustard anticancer agent.
[0013] The present invention also provides a therapeutic agent for
treatment of myeloma comprising anti-IL-6 receptor antibody for use
in combination with mechlorethamine, nitrogen mustard N-oxide,
melphalan, uramustin, ifosfamide, chlorambucil, or
cyclophosphamide.
[0014] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a reshaped human PM-1 antibody for
use in combination with melphalan.
[0015] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a nitrogen mustard anticancer agent
for use in combination with anti-IL-6 receptor antibody.
[0016] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a nitrogen mustard anticancer agent
for use in combination with anti-IL-6 receptor monoclonal
antibody.
[0017] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a nitrogen mustard anticancer agent
for use in combination with PM-1 antibody.
[0018] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a nitrogen mustard anticancer agent
for use in combination with a reshaped human PM-1 antibody.
[0019] The present invention also provides a therapeutic agent for
treatment of myeloma comprising mechlorethamine, nitrogen mustard
N-oxide, melphalan, uramustin, ifosfamide, chlorambucil, or
cyclophosphamide in combination with anti-IL-6 receptor
antibody.
[0020] The present invention also provides a therapeutic agent for
treatment of myeloma comprising melphalan for use in combination
with a reshaped human PM-1 antibody.
[0021] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a nitrogen mustard anticancer agent
and anti-IL-6 receptor antibody.
[0022] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a nitrogen mustard anticancer agent
and anti-IL-6 receptor monoclonal antibody.
[0023] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a nitrogen mustard anticancer agent
and PM-1 antibody.
[0024] The present invention also provides a therapeutic agent for
treatment of myeloma comprising a nitrogen mustard anticancer agent
and a reshaped human PM-1 antibody.
[0025] The present invention also provides a therapeutic agent for
treatment of myeloma comprising mechlorethamine, nitrogen mustard
N-oxide, melphalan, uramustin, ifosfamide, chlorambucil, or
cyclophosphamide and anti-IL-6 receptor antibody.
[0026] The present invention provides a therapeutic agent for
treatment of myeloma comprising melphalan and a reshaped human PM-1
antibody.
BRIEF EXPLANATION OF DRAWINGS
[0027] FIG. 1 is a graph showing the relationship of anti-human
IL-6 receptor antibody concentration and melphalan concentration
with the growth (incorporation of .sup.3H-labeled thymidine) of a
human myeloma cell line in the presence of 0.1 ng/ml IL-6.
[0028] FIG. 2 is a graph showing the relationship of anti-human
IL-6 receptor antibody concentration and melphalan concentration
with the growth (incorporation of .sup.3H-labeled thymidine) of a
human myeloma cell line in the presence of 1 ng/ml IL-6.
[0029] FIG. 3 is a graph showing the relationship of anti-human
IL-6 receptor antibody concentration and adriamycin concentration
with the growth (incorporation of .sup.3H-labeled thymidine) of a
human myeloma cell line in the presence of 0.1 ng/ml IL-6.
[0030] FIG. 4 is a graph showing the relationship of anti-human
IL-6 receptor antibody concentration and adriamycin concentration
with the growth (incorporation of .sup.3H-labeled thymidine) of a
human myeloma cell line in the presence of 1 ng/ml IL-6.
[0031] FIG. 5 is a graph showing the relationship of anti-human
IL-6 receptor antibody concentration and vincristine concentration
with the growth (incorporation of .sup.3H-labeled thymidine) of a
human myeloma cell line in the presence of 0.1 ng/ml IL-6.
[0032] FIG. 6 is a graph showing the relationship of anti-human
IL-6 receptor antibody concentration and vincristine concentration
with the growth (incorporation of .sup.3H-labeled thymidine) of a
human myeloma cell line in the presence of 1 ng/ml IL-6.
[0033] FIG. 7 is a graph showing the survival days of the mice
implanted with human myeloma cells in a single-drug administration
(1 mg/kg) of anti-human IL-6 receptor antibody (hPM-1) and
melphalan, or in a combined use thereof.
[0034] FIG. 8 is a graph showing the amount of M protein in the
mice implanted with human myeloma cells in a single-drug
administration (1 mg/kg) of anti-human IL-6 receptor antibody
(hPM-1) and melphalan, or in a combined use thereof.
[0035] FIG. 9 is a graph showing the survival days of mice
implanted with human myeloma cells in a single-drug administration
(3 mg/kg) of anti-human IL-6 receptor antibody (hPM-1)
and-melphalan, or in a combined use thereof, with a synergistic
effect obtained by the combined use.
[0036] FIG. 10 is a graph showing the changes in body weight of
mice implanted with human myeloma cells in a single-drug
administration of anti-human IL-6 receptor antibody (hPM-1) and
melphalan, or in a combined use thereof.
[0037] FIG. 11 is a graph showing the amount of M protein in the
serum of mice implanted with human myeloma cells at 30 days after
the tumor implantation in the anti-human IL-6 receptor antibody
(hPM1) single-drug administration group and in the melphalan
single-drug administration group.
[0038] FIG. 12 is a graph showing the amount of M protein in the
serum of mice implanted with human myeloma cells at 35 days after
the tumor implantation in the melphalan single-drug administration
group and the anti-human IL-6 receptor antibody (hPM1)-combined
administration group.
[0039] FIG. 13 is a graph showing the amount of M protein in the
serum of mice implanted with human myeloma cells at 42 days after
the tumor implantation in the melphalan single-drug administration
group and the anti-human IL-6 receptor antibody (hPM1)-combined
administration group.
[0040] FIG. 14 is a graph showing the survival period by a survival
curve of mice implanted with human myeloma cells in the melphalan
single-drug administration group and the anti-human IL-6 receptor
antibody (hPM1)-combined administration group, indicating an
enhanced effect by combined use.
[0041] FIG. 15 is a graph showing the changes in body weight of
mice implanted with human myeloma cells in the anti-human IL-6
receptor antibody single-drug administration group.
[0042] FIG. 16 is a graph showing the changes in body weight of
mice implanted with human myeloma cells in the melphalan
single-drug administration group and the anti-human IL-6 receptor
antibody (hPM1)-combined administration group.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0043] Nitrogen mustard anticancer agents for use in the present
invention is a general term for the anticancer agents having a
partial structure called nitrogen mustard having the structure:
[0044] examples of which agents include:
[0045] Mechlorethamine,
[0046] Nitrogen mustard N-oxide (methyl-bis(.beta.-chloroethyl)
amine N-oxide hydrochloride),
[0047] Melphalan (p-[bis(2-chloroethyl)amino-L-phenylalanine),
[0048] Chlorambucil (p-bis(2-chloroethyl)amino-phenylbutyric
acid),
[0049] Uramustin (5-bis(2-chloroethyl)aminouracil),
[0050] Ifosmide(N,N'-bis(2-chloroethyl)-N', O-propylenephosphoric
acid ester diamide
[0051] Cyclophosphamide(N,N'-bis(.beta.-chloroethyl)-N',
O-propylenephosphoric acid ester diamide,
[0052] and the like.
[0053] In accordance with the present invention, these nitrogen
mustard anticancer agents may be used as a single drug or in
combination. Among them, melphalan is also called sarcolysine or
L-phenylalanine mustard and has the following structure: 1
[0054] Mechlorethamine can be obtained by a known method, for
example, a method described in Abrams et al., J. Soc. Chem. Ind.
(London) (1949) 68, 280.
[0055] Nitrogen mustard N-oxide can be obtained by a known method,
for example, a method described in Aiko et al., J. Pharm. Soc.
Japan (1952) 72, 1297.
[0056] Melphalan can be obtained by a known method, for example, a
method described in Bergel, F. et al., J. Chem. Soc. (1954)
2409.
[0057] Chlorambucil can be obtained by a known method, for example,
a method described in Balazc, M. K. et al., J. Pharm. Sci. (1970)
59, 563.
[0058] Uramustin can be obtained by a known method, for example, a
method described in Lyttle and Petering, J. Am. Chem. Soc. (1958)
80, 6459.
[0059] Ifosfamide can be obtained by a known method, for example, a
method described in Arnold H. et al., U.S. Pat. No. 3,732,340,
(1973 to Asta) or Brassfield, HJ. A. et al., J. Am. Che. Soc.
(1975) 97, 4143.
[0060] Cyclophosphamide can be obtained by a known method, for
example, a method described in Arnold H. et al., Angew. Chem.
(1958) 70, 539.
[0061] 1. Anti-IL-6 Receptor Antibody
[0062] Anti-IL-6 receptor antibodies for use in the present
invention may be of any origin, any kind (monoclonal or
polyclonal), and any form, as long as they have a higher
therapeutic effect for myeloma when administered in combination
with a nitrogen mustard anticancer agent than when an anti-IL-6
receptor antibody alone is administered or when a nitrogen mustard
anticancer agent alone is administered.
[0063] Anti-IL-6 receptor antibodies for use in the present
invention can be obtained as polyclonal or monoclonal antibodies
using a known method. As the anti-IL-6 receptor antibodies for use
in the present invention, monoclonal antibodies of, in particular,
a mammalian origin, are preferred. Monoclonal antibodies of a
mammalian origin include those produced by a hybridoma and
recombinant antibody produced by a host which has been transformed
with an expression vector containing genetically engineered
antibody genes. Anti-IL-6 receptor antibodies for use in the
present invention, via binding to IL-6 receptor, block the binding
of IL-6 to IL-6 receptor, and thereby inhibit signal transmission
of IL-6, and therefore are antibodies which inhibit the biological
activity of IL-6.
[0064] Examples of such antibodies include PM-1 antibody (Hirata,
et al., J. Immunology (1989) 143, 2900-2906), or AUK12-20 antibody,
AUK64-7 antibody or AUK146-15 antibody (International Patent
Application WO 92-19759), and the like. Of them, PM-1 antibody is
most preferred.
[0065] Incidentally, the hybridoma cell line which produces PM-1
antibody has been internationally deposited under the provisions of
the Budapest Treaty as PM-1 on Jul. 10, 1990 with the National
Institute of Bioscience and Human Technology, Agency of Industrial
Science and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi,
Ibaraki, Japan, as FERM BP-2998.
[0066] 2. Antibody Produced by Hybridoma
[0067] Monoclonal antibodies can be obtained by constructing a
hybridoma using basically a known procedure as described bellow.
Thus, IL-6 receptor is used as an immunizing antigen and is
immunized in the conventional method of immunization. The immune
cells thus obtained are fused with known parent cells in the
conventional cell fusion process, and then screened by the
conventional screening method to screen monoclonal
antibody-producing cells.
[0068] Specifically, monoclonal antibodies may be obtained in the
following manner.
[0069] For example, IL-6 receptor used as the immunizing antigen
for obtaining antibody is not limited to any animal species, but
IL-6 receptor derived from humans is particularly preferred. For
human IL-6 receptor, IL-6 receptor protein can be obtained using a
gene sequence disclosed in European Patent Application EP 325474.
There are two kinds of IL-6 receptor: IL-6 receptor expressed on
the cell membrane, and IL-6 receptor detached from the cell
membrane (Soluble IL-6 Receptor; Yasukawa et al., J. Biochem.
(1990) 108, 673-676).
[0070] Soluble IL-6 receptor is composed mainly of the
extracellular region of IL-6 receptor bound to the cell membrane,
and soluble IL-6 receptor is different from the membrane-bound IL-6
receptor in that the former lacks the transmembrane region or both
of the transmembrane region and the intracellular region. In
accordance with the present invention, IL-6 receptor used as the
immunizing antigen may be either the membrane-bound or the soluble
IL-6 receptor. Alternatively, it may be a mutant thereof.
[0071] After a gene encoding IL-6 receptor is inserted into a known
expression vector to transform an appropriate host cell, the
desired IL-6 receptor protein is purified from the host cell or a
culture supernatant thereof using a known method, and the IL-6
receptor protein thus purified may be used as the immunization
antigen. Alternatively, cells that express IL-6 receptor protein
may be used as the immunization antigen.
[0072] Preferably mammals to be immunized with the immunization
antigen are selected in consideration of their compatibility with
the parent cells for use in cell fusion and they generally include,
but are not limited to, rodents, logomorphas, and primates.
[0073] As rodents, for example, mice, rats, hamsters, etc. are
used. As logomorphas, for example, rabbits are used. As primates,
for example, monkeys are used. As monkeys, catarrhines (Old-World
monkeys) such as cynomolgi (crab-eating macaque), rhesus monkeys,
sacred baboons, chimpanzees etc. are used.
[0074] Immunization of animals with an immunization antigen is
carried out using a known method. A general method, for example,
involves intraperitoneal or subcutaneous administration of an
immunization antigen to the mammal. Specifically, an immunization
antigen which was diluted and suspended in an appropriate amount of
phosphate buffered saline (PBS) or physiological saline etc. is
mixed with an appropriate amount of Freund's complete adjuvant.
After being emulsified, it is preferably administered to a mammal
for several times every 4 to 21 days. Additionally a suitable
carrier may be used at the time of immunization of the immunization
antigen.
[0075] After the immunization and confirmation of the increase in
the desired antibody levels in the serum by a conventional method,
immune cells are taken out from the mammal and are subjected to
cell fusion, in which especially preferred immune cells are the
spleen cells.
[0076] The mammalian myeloma cells as the other parent cells which
are subjected to cell fusion with the above-mentioned immune cells
preferably include various known cell lines such as P3
(P3.times.63Ag8.653) (Kearney, J. F. et al., J. Immunol. (1979)
123, 1548-1550), P3.times.63Ag8U.multidot.U1 (Yelton, D. E. et al.,
Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1
(Kohler, G. and Milstein, C., Eur. J. Immunol. (1976) 6, 511-519),
MPC-11 (Margulies, D. H. et al., Cell (1976) 8, 405-415), SP2/0
(Shulman, M. et al., Nature (1978)-276, 269-270), FO (de St. Groth,
S. F. and Scheidegger, D., J. Immunol. Methods (1980) 35, 1-21),
S194 (Trowbridge, I. S., J. Exp. Med. (1978) 148, 313-323), R210
(Galfre, G. et al., Nature (1979) 217, 131-133) and the like.
[0077] Cell fusion between the above immune cells and the myeloma
cells may be essentially conducted in accordance with a known
method such as is described in Milstein et al. (Galfre, G. and
Milstein, C., Methods Enzymol. (1981) 73, 3-46) and the like.
[0078] More specifically, the above cell fusion is carried out in
the conventional nutrient broth in the presence of, for example, a
cell fusion accelerator. As the cell fusion accelerator, for
example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like
may be used, and an adjuvant such as dimethyl sulfoxide etc. may be
added as desired to enhance efficiency of the fusion.
[0079] The preferred ratio of the immune cells and the myeloma
cells for use is, for example, 1 to 10 times more immune cells than
the myeloma cells. Examples of culture media to be used for the
above cell fusion include RPMI 1640 medium and MEM culture medium
suitable for the growth of the above myeloma cell lines, and the
conventional culture medium used for this type of cell culture, and
besides a serum supplement such as fetal calf serum (FCS) may be
added.
[0080] In cell fusion, predetermined amounts of the above immune
cells and the myeloma cells are mixed well in the above culture
liquid, to which a PEG solution previously heated to about
37.degree. C., for example a PEG solution with a mean molecular
weight of 1000 to 6000, is added at a concentration of 30 to 60%
(w/v) and mixed to obtain the desired fusion cells (hybridomas).
And then by repeating a sequential addition of a suitable culture
liquid and centrifugation to remove the supernatant, cell fusion
agents etc. that are undesirable for the growth of the hybridoma
can be removed.
[0081] Said hybridoma is selected by culturing in the conventional
selection medium, for example, HAT culture medium (a culture liquid
containing hypoxanthine, aminopterin, and thymidine). Culturing in
said HAT culture medium is continued generally for the period of
time sufficient to effect killing of the cells other than the
desired hybridoma (non-fusion cells), generally several days to
several weeks. The conventional limiting dilution method is
conducted in which the hybridomas producing the desired antibody
are screened and monoclonally cloned.
[0082] In addition to obtaining the above hybridoma by immunizing
an animal other than the human with an antigen, it is also possible
to immunize human lymphocytes in vitro with IL-6 receptor protein
or IL-6 receptor protein-expressing cells, and the resulting
immunized lymphocytes are fused with a myeloma cell, for example
U266, having the ability of dividing permanently to obtain a
hybridoma that produces the desired human antibody having the
activity of binding to and neutralizing IL-6 receptor (Japanese
Post-examined Patent Publication (Kokoku) 1-59878). Furthermore, a
transgenic animal having a repertoire of human antibody genes is
immunized with the antigen IL-6 receptor or IL-6
receptor-expressing cells to obtain anti-IL-6 receptor
antibody-producing cells. The cells are then fused with myeloma
cells to obtain hybridomas that are used to obtain human antibody
to IL-6 receptor (see International Patent Application WO 92-03918,
WO 93-12227, WO 94-02602, WO 94-25585, WO 96-33735 and WO
96-34096).
[0083] The monoclonal antibody-producing hybridomas thus
constructed can be maintained in the conventional culture liquid,
or can be stored for a prolonged period of time in liquid
nitrogen.
[0084] In order to obtain monoclonal antibodies from said
hybridoma, there can be mentioned a method in which said hybridoma
is cultured in the conventional method and the antibodies are
obtained as the supernatant, or a method in which the hybridoma is
transplanted to and grown in a mammal compatible with said
hybridoma and the antibodies are obtained as the ascites. The
former method is suitable for obtaining highly purified antibodies,
whereas the latter is suitable for a large scale production of
antibodies.
[0085] In addition to using a hybridoma for antibody production,
immune cells such as antibody-producing immunized lymphocytes which
has been immortalized with an oncogene can be used.
[0086] 3. Recombinant Antibody
[0087] Monoclonal antibodies may be also obtained as a recombinant
antibody which has been produced by the recombinant gene
technology. For example, recombinant antibody can be obtained by
cloning a gene of an antibody from a hybridoma or an immune cell
such as antibody-producing immunized lymphocytes, and then
integrated into a suitable vector, which is then introduced into a
host to produce said antibody (see, for example, Borrebaeck, C. A.
K. and Larrick, J. W., THERAPEUTIC MONOCLONAL ANTIBODIES, published
in the United Kingdom by MACMILLAN PUBLISHERS LTD. 1990).
[0088] Specifically, mRNA encoding the variable region (V region)
of anti-IL-6 receptor antibody is isolated from the hybridoma
producing anti-IL-6 receptor antibody. The isolation of mRNA is
conducted by preparing total RNA using, for example, a known method
such as the guanidine ultracentrifuge method (Chirgwin, J. M. et
al., Biochemistry (1979) 18, 5294-5299), the AGPC method
(Chomczynski, P. and Sacchi, N., Anal. Biochem. (1987) 162,
156-159), and then mRNA is purified from the total RNA using the
mRNA Purification kit (Pharmacia) and the like. Alternatively, mRNA
can be directly prepared using the Quick Prep mRNA Purification Kit
(Pharmacia).
[0089] cDNA of the V region of antibody may be synthesized from the
mRNA thus obtained using a reverse transcriptase. cDNA may be
synthesized using the AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit (Seikagaku Kogyo), and the like. Alternatively, for
the synthesis and amplification of cDNA, the 5'-Ampli FINDER RACE
Kit (Clontech) and the 5'-RACE method (Frohman, M. A. et al., Proc.
Natl. Acad. Sci. U.S.A. (1988) 85, 8998-9002; Belyavsky, A. et al.,
Nucleic Acids Res. (1989) 17, 2919-2932) which employs polymerase
chain reaction (PCR) may be used.
[0090] The desired DNA fragment is purified from the PCR product
obtained and may be ligated to vector DNA. Moreover, a recombinant
vector is constructed therefrom and then is transfected into E.
coli etc., which is selected to prepare the desired recombinant
vector. The base sequence of the desired recombinant vector may be
confirmed by a known method such as the dideoxy nucleotide chain
termination method.
[0091] Once the DNA encoding the V region of the desired anti-IL-6
receptor antibody has been obtained, it may be ligated to DNA
encoding the constant region (C region) of the desired antibody,
which is then integrated into an expression vector. Alternatively,
DNA encoding the V region of the antibody may be integrated into an
expression vector which already contains DNA encoding the C region
of the antibody. The C region of the antibody may be derived from
the same animal species as that of the V region, or from a
different animal species from that of the V region.
[0092] In order to produce anti-IL-6 receptor antibody for use in
the present invention, the antibody gene is integrated into an
expression vector so as to be expressed under the control of an
expression regulatory region, for example an enhancer and/or a
promoter. Subsequently, the expression vector is transformed into a
host cell and the antibody is then expressed therein.
[0093] The antibody gene may be expressed by integrating separately
DNA encoding a heavy chain (H chain) and a light chain (L chain) of
the antibody into an expression vector and co-transforming the host
cell, or by integrating DNA encoding an H chain and an L chain into
a single expression vector and transforming the host cell
(International Patent Application WO 94-11523).
[0094] 4. Altered Antibody
[0095] As recombinant antibodies for use in the present invention,
artificially altered recombinant antibodies such as chimeric
antibody and humanized antibody can be used for the purpose of
lowering xenogenic antigenicity against humans. Altered antibodies
can have the C regions of human antibody and antibodies such as
chimeric antibody or humanized antibody can be used. These altered
antibodies can be produced using known methods.
[0096] Chimeric antibody can be obtained by ligating the thus
obtained DNA encoding the V region of antibody other than human
antibody to DNA encoding the C region of human antibody, which is
then integrated into an expression vector and introduced into a
host for production of the antibody therein (see European Patent
Application EP 125023, and International Patent Application WO
92-19759,). Using this known method, chimeric antibody useful for
the present invention can be obtained.
[0097] Plasmid coding for the V region of the L chain or the V
region of the H chain of PM-1 antibody has each been designated as
pPM-k3 and pPM-h1, respectively, and E. coli having a respective
plasmid has been internationally deposited under the provisions of
the Budapest Treaty as NCIMB40366 and NCIMB40362 on Feb. 11, 1991
with the National Collections of Industrial and Marine Bacteria
Limited.
[0098] Humanized antibody which is also called reshaped human
antibody has been made by transplanting the complementarity
determining regions (CDRs) of an antibody of a mammal other than
the human, for example mouse antibody, into the CDRs of human
antibody. The general recombinant DNA technology for preparation of
such antibodies is also known (see European Patent Application EP
125023 and International Patent Application WO 92-19759).
[0099] Specifically, a DNA sequence which was designed to ligate
the CDRs of mouse antibody with the framework regions (FRs) of
human antibody is synthesized from several divided oligonucleotides
having sections overlapping with one another at the ends thereof,
and the oligonucleotides are then synthesized into one integrated
DNA. The DNA thus obtained is ligated to a DNA encoding a C region
of human antibody and then is integrated into an expression vector,
which is introduced into a host for antibody production (see
European Patent Application EP 239400 and International Patent
Application WO 92-19759).
[0100] For the FRs of human antibody being ligated to CDRS, the FR
that make CDR a favorable antigen-binding site is selected. When
desired, Amino acids in the FR of antibody V region may be
substituted so that the CDR of humanized antibody may form an
appropriate antigen binding site (Sato, K. et al., Cancer Res.
(1993) 53, 851-856).
[0101] A preferred embodiment of humanized antibody for use in the
present invention includes humanized PM-1 antibody (see
International Patent Application WO 92-19759). In the humanized
PM-1 antibody, CDRs of the PM-1 antibody derived from a mouse have
been ligated to the FRs of the human antibody REI for the L chain,
and the FRs of the human antibody NEW, and part of the amino acid
residues of the FR has been substituted to obtain antigen-binding
activity.
[0102] In order to produce anti-IL-6 receptor antibody for use in
the present invention, the antibody gene is integrated into an
expression vector so as to be expressed under the control of an
expression regulatory region, for example an enhancer and/or a
promoter. Subsequently, the expression vector is transformed into a
host cell and the antibody is then expressed therein.
[0103] The antibody gene may be expressed by integrating separately
DNAs encoding a heavy chain (H chain) and a light chain (L chain)
of an antibody into an expression vector and co-transforming the
host cell, or by integrating a DNA encoding an H chain and an L
chain into a single expression vector and transforming the host
cell (International Patent Application WO 94-11523).
[0104] Chimeric antibody consists of the V regions of antibody
derived from a mammal other than the human and the C regions
derived from human antibody, whereas humanized antibody consists of
the CDRs of antibody derived from a mammal other than the human and
the FRs and the C regions of antibody derived from human antibody.
Accordingly, since the amino acid sequences derived from a mammal
other than the human are reduced to a minimum in the above
antibodies, antigenicity thereof in the human body is reduced so
that they are useful as the active ingredient of the therapeutic
agents of the present invention.
[0105] As the C region of human antibody, there can be used, for
example, C.gamma.1, C.gamma.2, C.gamma.3, or C.gamma.4. The C
region of a human antibody may also be modified in order to improve
the stability of antibody or of the production thereof.
[0106] 5. Antibody Fragments and Modified Antibody
[0107] Antibodies for use in the present invention may be fragments
of antibody or modified versions thereof as long as they bind to
IL-6 receptor and thereby inhibit the binding of IL-6 and IL-6
receptor to block signal transmission and to inhibit the biological
activity of IL-6. They are antibody fragments or modified
antibodies which, when used in combination with a nitrogen mustard
anticancer agent, have a higher therapeutic effect for myeloma than
IL-6 receptor antibody alone or a nitrogen mustard anticancer agent
alone.
[0108] For example, as fragments of antibody, there may be
mentioned Fab, F(ab')2, Fv or single-chain Fv (scFv) in which Fv's
of H chain and L chain were ligated via a suitable linker.
Specifically antibodies are treated with an enzyme, for example,
papain or pepsin, to produce antibody fragments, or genes encoding
these antibody fragments are constructed, and then integrated into
an expression vector, which is expressed in a suitable host cell
(see, for example, Co, M. S. et al., J. Immunol. (1994) 152,
2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989)
178, 476-496; Plucktrun, A. and Skerra, A., Methods Enzympl. (1989)
178, 497-515; Lamoyi, E., Methods Enzymol. (1986.) 121, 652-663;
Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird,
R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).
[0109] scFv can be obtained by ligating a V region of an H chain
and a V region of an L chain of an antibody. In the scFv, the V
region of H chainland the V region of L chain are preferably
ligated via a linker, preferably a peptide linker (Huston, J. S. et
al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The V
region of H chain and the V region of L chain in the scFv may be
derived from any of the above-mentioned antibodies. As the peptide
linker for ligating the V regions, any single-chain peptide
comprising, for example, 12-19 amino acid residues may be used.
[0110] DNA encoding scFv can be obtained using a DNA encoding an H
chain or a V region of an H chain of the above antibody and a DNA
encoding an L chain or a V region of an L chain of the above
antibody as the template by amplifying the portion of the DNA
encoding the desired amino acid sequence among the above sequences
by the PCR technique with the primer pair specifying the both ends
thereof, and by further amplifying the combination of DNA encoding
the peptide linker portion and the primer pair which defines that
both ends of said DNA be ligated to the H chain and the L chain,
respectively.
[0111] Once DNAs encoding scFv are constructed, an expression
vector containing them and a host transformed with said expression
vector can be obtained by the conventional methods, and scFv can be
obtained using the resultant host by the conventional methods.
[0112] Antibody fragments may be those antibody fragments part of
which sequence has undergone mutation, substitution, deletion, or
insertion. These antibody fragments can be produced by obtaining
the gene thereof in a similar manner to that mentioned above and by
allowing it to be expressed in a host. "Antibody" as used in the
claim of the present application encompasses these antibody
fragments.
[0113] As modified antibodies, anti-IL-6 receptor antibody
associated with various molecules such as polyethylene glycol (PEG)
can be used. "Antibody" as used in the claim of the present
application encompasses these modified antibodies. These modified
antibodies can be obtained by chemically modifying the antibodies
thus obtained. These methods have already been established in the
art.
[0114] 6. Expression and Production of Recombinant Antibody,
Altered Antibody, and Antibody Fragment
[0115] Antibody genes constructed as mentioned above may be
expressed and obtained in a known manner. In the case of mammalian
cells, expression may be accomplished using an expression vector
containing a commonly used useful promoter, an antibody gene to be
expressed, and DNA in which the poly A signal has been operably
linked at 3' downstream thereof. Examples of the promoter/enhancer
include human cytomegalovirus immediate early
promoter/enhancer.
[0116] Additionally, as the promoter/enhancer which can be used for
expression of antibody for use in the present invention, there can
be used viral promoters/enhancers such as retrovirus, polyoma
virus, adenovirus, and simian virus 40 (SV40), and
promoters/enhancers derived from mammalian cells such as human
elongation factor 1.alpha. (HEF1.alpha.).
[0117] For example, expression may be readily accomplished by the
method of Mulligan, R. C. et al. (Nature (1979) 277, 108-114) when
SV40 promoter/enhancer is used, and by the method of Mizushima, S.
et al. (Nucleic Acids Res. (1990) 18, 5322) when HEF1.alpha.
promoter/enhancer is used.
[0118] In the case of E. coli, expression may be conducted by
operably linking a commonly used promoter, a signal sequence for
antibody secretion, and an antibody gene to be expressed, followed
by expression thereof. As the promoter, for example, there can be
mentioned lacz promoter and araB promoter. The method of Ward, E.
S. et al. (Nature (1989) 341, 544-546; FASEB J. (1992) 6,
2422-2427) may be used when lacz promoter is used, and the method
of Better, M. et al. (Science (1988) 240, 1041-1043) may be used
when araB promoter is used.
[0119] As a signal sequence for antibody secretion, when produced
in the periplasm of E. coli, the pelB signal sequence (Lei, S. P.
et al., J. Bacteriol. (1987) 169, 4379-4383) can be used. After
separating the antibody produced in the periplasm, the structure of
the antibody is appropriately refolded before use (see, for
example, International Patent Application WO 96-30394).
[0120] As the origin of replication, there can be used those
derived from SV40, polyoma virus, adenovirus, bovine papilloma
virus (BPV), and the like. Furthermore, for gene amplification in
the host cell system, expression vectors can include as selection
markers the aminoglycoside transferase (APH) gene, the thymidine
kinase (TK) gene, E. coli xanthine guaninephosphoribosyl
transferase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene,
and the like.
[0121] For the production of antibody for use in the present
invention, any production system can be used, and the production
system of antibody preparation comprises the in vitro or the in
vivo production system.
[0122] As the in vitro production system, there can be mentioned a
production system which employs eukaryotic cells and the production
system which employs prokaryotic cells.
[0123] When eukaryotic cells are used, there are the production
systems which employ animal cells, plant cells, and fungal cells.
Known animal cells include (1) mammalian cells such as CHO cells,
COS cells, myeloma cells, baby hamster kidney (BHK) cells, HeLa
cells, and Vero cells, (2) amphibian cells such as Xenopus oocytes,
or (3) insect cells such as sf9, sf21, and Tn5. Known plant cells
include, for example, those derived from the Nicotiana family, more
specifically cells derived from Nicotiana tabacum which is
subjected to callus culture. Known fungal cells include (1) yeasts
such as the Saccharomyces family, more specifically Saccharomyces
cereviceae, or (2) mold fungi such as the genus Aspergillus, more
specifically Aspergillus niger.
[0124] When prokaryotic cells are used, there are the production
systems which employ bacterial cells. Known bacterial cells include
Escherichia coli, and Bacillus subtilis.
[0125] By introducing via transformation the gene of the desired
antibody into these cells and culturing the transformed cells in
vitro, the antibody can be obtained. Culturing is conducted in the
known methods. For example, as the culture liquid for mammalian
cells, DMEM, MEM, RPMI1640, IMDM and the like can be used, and
serum supplements such as fetal calf serum (FCS) may be used in
combination. In addition, antibodies may be produced in vivo by
implanting cells into which the antibody gene has been introduced
into the peritoneal cavity of an animal, and the like.
[0126] As in vivo production systems, there can be mentioned those
which employ animals and those which employ plants. When animals
are used, there are the production systems which employ mammals and
insects.
[0127] As mammals, goats, pigs, sheep, mice, and cattle can be used
(Glaster, V., SPECTRUM Biotechnology Applications, 1993).
[0128] When mammals are used, transgenic animals can be used. For
example, antibody genes are inserted into the gene encoding protein
which is inherently produced in the milk such as goat .beta. casein
to prepare fusion genes. DNA fragments containing the fusion gene
into which the antibody gene has been inserted are injected to a
goat embryo, and the embryo is introduced into a female goat. The
desired antibody is obtained from the milk produced by the
transgenic goat borne to the goat who received the embryo or
offsprings thereof. In order to increase the amount of milk
produced containing the desired antibody produced by the transgenic
goat, hormones may be given to the transgenic goat as appropriate.
(Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
[0129] Also as insects, silkworms can be used. When silkworms are
used, baculovirus into which the desired antibody gene has been
inserted is infected to the silkworm, and the desired antibody can
be obtained from the body fluid of the silkworm (Maeda, S. et al.,
Nature (1985) 315, 592-594).
[0130] Moreover, when plants are used, tabacco, for example, may be
used. When tabacco is used, the desired antibody gene is inserted
into an expression vector for plants, for example pMON 530, and
then the vector is introduced into a bacterium such as
Agrobacterium tumefaciens. The bacterium is then infected to
tobacco such as Nicotiana tabacum to obtain the desired antibody
from the leaves of the tobacco (Ma, J. K. et al., Eur. J. Immunol.
(1994) 24, 131-138).
[0131] An antibody gene is introduced, as mentioned above, into
these animals or plants, and then the antibody is produced in such
animals and plants and is collected therefrom.
[0132] When antibody is produced in in vitro or in vivo production
systems, as mentioned above, DNAs encoding an H chain and an L
chain of an antibody are separately integrated into expression
vectors and the hosts are transformed simultaneously, or a DNA
encoding an H chain and an L chain of an antibody is integrated
into a single expression vector and the host is transformed
therewith (International Patent Application WO 94-11523).
[0133] 7. Separation and Purification of Antibody
[0134] Antibodies expressed and produced as described above can be
separated from inside or outside of the host cell and then may be
purified to homogeneity. Separation and purification of antibody
for use in the present invention may be accomplished by methods of
separation and purification conventionally used for proteins
without any limitation.
[0135] For example, separation and purification of antibody may be
accomplished by combining, as appropriate, column chromatography
such as affinity chromatography, filtration, ultracentrifugation,
salting-out, dialysis and the like (Antibodies: A Laboratory
Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory,
1988).
[0136] As the column used for affinity chromatography, there can be
mentioned Protein A column and Protein G column. Examples of the
column employing Protein A column are Hyper D, POROS, Sepharose F.
F. (Pharmacia) and the like.
[0137] Chromatography other than affinity chromatography includes,
for example, ion exchange chromatography, hydrophobic
chromatography, gel-filtration, reverse-phase chromatography,
absorption chromatography and the like (Strategies for Protein
Purification and Characterization: A Laboratory Course Manual, Ed
Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press,
1996). Furthermore, said chromatography may be carried out using a
liquid-phase chromatography such as HPLC, FPLC, and the like.
[0138] 8. Measurement of Antibody Concentration
[0139] The concentration of antibody obtained as above can be
determined by measurement of absorbance or by the enzyme-linked
immunosorbent assay (ELISA) and the like. Thus, when absorbance
measurement is employed, the antibody obtained is appropriately
diluted with PBS and then the absorbance is measured at 280 nm,
followed by calculation using the absorption coefficient of, though
different with species and subclasses, 1.4 OD at 1 mg/ml in the
case of human antibody.
[0140] When the ELISA method is used, measurement is conducted as
follows. Thus, 100 .mu.l of goat anti-human IgG antibody diluted to
1 .mu.g/ml in 0.1 M bicarbonate buffer, pH 9.6, is added to a
96-well plate (Nunc), and is incubated overnight at 4.degree. C. to
immobilize the antibody. After blocking, 100 .mu.l each of
appropriately diluted antibody of the present invention or samples
containing the antibody, or 100 .mu.l of human IgG of a known
concentration as the concentration standard is added, and incubated
at room temperature for 1 hour.
[0141] After washing, 100 .mu.l of 5000-fold diluted alkaline
phosphatase-labeled anti-human IgG antibody is added, and incubated
at room temperature for 1 hour. After washing, the substrate
solution is added and incubated, followed by measurement of
absorbance at 405 nm using the MICROPLATE READER Model 3550
(Bio-Rad) to calculate the concentration of the desired antibody
based on the absorbance of the concentration standard IgG.
[0142] For determination of antibody concentration, BIAcore
(Pharmacia) can be used.
[0143] 9. Confirmation of the Activity of Antibody
[0144] Evaluation of activity of anti-IL-6 receptor antibody of the
present invention can be conducted using a commonly known method.
IL-6 is added to a plate in which IL-6 responsive cells such as
HN60.BSF2 cells were cultured. Then, evaluation is made in the
presence of anti-IL-6 receptor antibody, using the incorporation of
.sup.3H labeled thymidine by IL-6 dependent cells as an index.
[0145] Alternatively, .sup.125I-labeled IL-6 and anti-IL-6 receptor
antibody are added to a plate in which IL-6 receptor-expressing
cells such as U266 have been cultured and then the amount of
.sup.125I-labeled IL-6 that is bound to the IL-6-expressing cells
is determined for evaluation (Antibodies: A Laboratory Manual. Ed
Harlow and David Lane, Cold Spring Harbor Laboratory, 1988).
[0146] As methods for determining the antigen-binding activity of
anti-IL-6 receptor antibody for use in the present invention, there
can be used ELISA, EIA (enzymeimmunoassay), RIA (radioimmunoassay),
or the fluorescent antibody method.
[0147] When ELISA is employed, for example, IL-6 receptor is added
to a 96-well plate onto which antibody against IL-6 receptor has
been immobilized, and then samples containing the desired anti-IL-6
receptor antibody, for example a culture supernatant of anti-IL-6
receptor antibody-producing cells or purified antibody, are added
thereto. Secondary antibody that recognizes the desired anti-IL-6
receptor antibody, labeled with an enzyme such as alkaline
phosphatase is added, and the plate is incubated, washed, and then
the enzyme substrate such as p-nitrophenyl phosphate is added
thereto. Then the absorbance is measured to evaluate the
antigen-binding activity. A soluble IL-6 receptor may be used as
the IL-6 receptor.
[0148] As methods for measuring the inhibition activity of ligand
receptor binding of the anti-IL-6 receptor antibody for use in the
present invention, the conventional Cell ELISA or the ligand
receptor binding assay can be used.
[0149] In the case of Cell ELISA, for example, cells expressing
IL-6 receptor are cultured in a 96-well plate and then immobilized
with paraformaldehyde etc. Alternatively, membrane fractions of
cells expressing IL-6 receptor are prepared and a 96-well plate on
which IL-6 receptors have been immobilized is prepared. To this are
added a sample containing the desired anti-IL-6 receptor antibody,
for example a culture supernatant of anti-IL-6 receptor
antibody-producing cells, and purified antibody, and IL-6 labeled
with a radioisotope such as .sup.125I, and then the plate is
incubated, washed, and radioactivity is measured to determine the
amount of IL-6 bound to the IL-6 receptor and thereby to evaluate
the inhibition activity of ligand receptor binding of anti-IL-6
receptor antibody.
[0150] In the inhibition assay of IL-6 binding to IL-6 receptor on
the cells, cells expressing IL-6 receptors are separated by means
of centrifugation etc. and resuspended to prepare a cell
suspension. A solution of IL-6 labeled with a radioisotope such as
.sup.125I, or a mixture of unlabeled IL-6 and labeled IL-6, and a
solution containing anti-IL-6 receptor antibody whose concentration
has been adjusted are added to the cell suspension. After
incubating for a certain period of time, the cells are separated,
and the radioactivity of the labeled IL-6 bound to the cell is
measured.
[0151] For evaluation of activity of the above antibody, BIAcore
(Pharmacia) can be used.
[0152] 10. Method of Administration and Pharmaceutical
Preparation
[0153] In accordance with the present invention, a nitrogen mustard
anticancer agent. and anti-IL-6 receptor antibody are used in
combination. "Used in combination" as used herein refers to a case
in which pharmaceutical compositions are administered at different
times, a case in which pharmaceutical compositions are administered
at the same time, and a case in which one pharmaceutical
composition comprising both of a nitrogen mustard anticancer agent
and anti-IL-6 receptor antibody is administered. In the former two
cases, a pharmaceutical composition comprising a nitrogen mustard
anticancer agent and a pharmaceutical composition comprising
anti-IL-6 receptor antibody may be given through the same
administration route, or a different administration route. Each of
these pharmaceutical compositions is given to cure or inhibit at
least partially the pathological symptoms of patients suffering
from diseases. The period of administration may be chosen, as
appropriate, depending on the age and conditions of the
patient.
[0154] Preferably, pharmaceutical compositions comprising anti-IL-6
receptor antibody may be administered parenterally, for example via
intravenous injection, drip infusion, intramuscular injection,
intraperitoneal injection, subcutaneous injection, and the like,
either systemically or locally. As local dosage-forms, preferably,
external preparations, local injections, and the like are used.
External preparations are chosen from liniments such as ointments,
gel, cream, emulsions, and liquids, tapes, plaster tapes such as
patches, or nebulas such as sprays and powders.
[0155] The effective dosage of anti-IL-6 receptor antibody is
chosen from the range of 0.001 mg to 1000 mg per kg of body weight
per day. Preferably, the dosage is selected from the range of 0.01
to 50 mg per body weight. The above doses depend on the
pathological conditions, and hence they are not limited to these
values. The number of administration is usually selected from, but
not limited to, once or twice per day, once per two to a few days,
or once per one to four weeks.
[0156] Pharmaceutical compositions comprising a nitrogen mustard
anticancer agent are preferably administered orally, but depending
on the nature of the active ingredient, the conditions of patients,
and the like, they may be given parenterally as well. For example
intravenous injection, drip infusion, intraarterial injection,
intramuscular injection, intratumor injection, intrathdracic
injection, or intraperitoneal injection, either systemically or
locally.
[0157] The effective dosage of nitrogen mustard anticancer agents
is different on their kind, but for melphalan, for example, oral
administration of 1 to 20 mg per day, every day or 1 to 6 times per
week, or as high-dose intravenous injection or infusion, single or
multiple doses of 20 to 200 mg/m.sup.2 is employed. For
cyclophosphamide, oral or intravenous administration of 50 to 2000
mg per dose usually for one to 5 times per week, to once per two
weeks to one month is employed. The number and the schedule of
administration are not limited to those mentioned above. Nitrogen
mustard anticancer agents may be given not only alone but also in
combination with vincristine, adriamycin, prednisolone, and the
like, as appropriate.
[0158] When a pharmaceutical composition comprising a nitrogen
mustard anticancer agent is administered simultaneously with
anti-IL-6 receptor antibody, the ratio, is, when combined with
daily oral administration of melphalan, 0.01 to 1000 fold (weight
ratio) relative to the dose of melphalan, though it is different on
the conditions of the patient and the administration schedule.
Alternatively a pharmaceutical composition comprising a certain
ratio of the two agents may be administered. However, as mentioned
above, the dose ratio varies with the conditions of the patient
etc., and hence it is not limited to the ratio mentioned above.
[0159] It is also possible to set up a schedule in which a
pharmaceutical composition comprising a nitrogen mustard anticancer
agent and anti-IL-6 receptor antibody are given at different time
points. For example, to patients for whom remission was introduced
by applying a nitrogen mustard anticancer agent or a combined
therapy including the agent as a constituent element, anti-IL-6
receptor antibody can be administered in order to maintain
remission. Furthermore, administration of a nitrogen mustard
anticancer agent or a combined therapy having the agent as a
constituent element and administration of anti-IL-6 receptor
antibody may be repeated every 1 to 4 weeks. For a nitrogen mustard
anticancer agent and anti-IL-6 receptor antibody, preferably the
former is given first, but the latter may be given first depending
on the conditions of the patient etc.
[0160] Pharmaceutical compositions of the present invention
comprising nitrogen mustard anticancer agents, pharmaceutical
compositions comprising anti-IL-6 receptor antibody, and
pharmaceutical compositions comprising a nitrogen mustard
anticancer agent and anti-IL-6 receptor antibody of the present
invention may contain pharmaceutically acceptable carriers and/or
additives depending on the route of administration.
[0161] Examples of such carriers or pharmaceutical additives
include water, a pharmaceutically acceptable organic solvent,
collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl
polymer, sodium carboxymethylcellulose, sodium, polyacrylate sodium
alginate, water-soluble dextran, sodium carboxymethyl starch,
pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic,
casein, gelatin, agar, diglycerin, glycerin, propylene glycol,
polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic
acid, human serum albumin (HSA), mannitol, sorbitol, lactose,
pharmaceutically acceptable surfactants and the like.
[0162] Actual additives are chosen from, but not limited to, the
above or combinations thereof depending on the dosage form of a
therapeutic agent of the present invention.
[0163] The present invention also encompasses a simultaneous or
sequential combined administration of a pharmaceutical agent of the
present invention with another agent, a biological agent, or a
synthetic agent. Other agents are selected from anti-inflammatory
agents, antiallergic agents, anti-platelet agents, other anticancer
agents or those that enhance or supplement the activity of the
object of the present invention.
EXAMPLES
[0164] The present invention will now be explained hereinbelow in
more detail with reference to the following reference examples,
experimental examples and working examples. It is to be noted that
the present invention is not limited to these examples in any
way.
Reference Example 1.
Construction of the Anti-IL-6 Receptor Antibody PM-1
[0165] The anti-IL-6 receptor antibody MT18 prepared by the method
of Hirata et al. (J. Immunol. (1989) 143, 2000-2006) was bound to
CNBr-activated Sepharose 4B (manufactured by Pharmacia Fine
Chemicals, Piscataway, N.J.) in accordance with the attached
directions to purify IL-6 receptor (Yamasaki et al., Science (1988)
241, 825-828).
[0166] Thus, a human myeloma cell line U266 was solubilized in 1 mM
p-paraaminophenylmethane sulphonylfluoride hydrochloride
(manufactured by Wako Chemicals) (digitonin buffer) containing 1%
digitonin (manufactured by Wako Chemicals), 10 mM triethanolamine
(pH 7.8), and 0.15M. NaCl, and then mixed with MT18 antibody
conjugated to Sepharose 4B beads. The beads were then washed six
times with the digitonin buffer to prepare partially purified IL-6
receptor to be used for immunization.
[0167] BALB/c mice were immunized four times every 10 days with the
above-mentioned partially purified IL-6 receptor obtained from
3.times.10.sup.9 U266 cells, and then hybridomas were prepared in a
conventional method. A culture supernatant of hybridomas from
growth-positive wells was evaluated for its ability of binding to
IL-6 receptor by the method described below. 5.times.10.sup.7 U266
cells were labeled with .sup.35S-methionine (2.5 mCi) and were
solubilized in the above digitonin buffer.
[0168] The solubilized U266 cells were mixed with 0.04 ml of MT18
antibody conjugated to Sepharose 4B beads and then washed six times
in the digitonin buffer. Using 0.25 ml of the digitonin buffer (pH
3.4), .sup.35S-methionine labeled IL-6 receptor was eluted, which
was neutralized with 0.025 ml of 1M Tris, pH 7.4. The hybridoma
culture supernatant 0.05 ml was mixed with 0.01 ml Protein G
Sepharose (manufactured by Pharmacia).
[0169] After washing, the Sepharose was incubated with 0.005 ml
solution of .sup.35S-labeled IL-6 receptor prepared above.
Immunoprecipitating substances were analyzed by SDS-PAGE to search
the culture supernatants of hybridoma that reacts with IL-6
receptor. As a result, a reaction-positive hybridoma clone PM-1 was
established. The anti-IL-6 receptor antibody PM-1 produced from the
hybridoma PM-1 had the IgG1 .kappa. subtype.
[0170] The activity of the antibody produced by the hybridoma PM-1
to inhibit the binding of IL-6 to IL-6 receptor was evaluated using
a human myeloma cell line U266. Recombinant human IL-6 was prepared
from E. coli (Hirano et al., Immunol. Lett. (1988) 17, 41), and was
labeled with .sup.125I using the Bolton-Hunter reagent (New England
Nclear, Boston, Mass.) (Taga et al., J. Exp. Med. (1987) 166,
967).
[0171] 4.times.10.sup.5 U266 cells were cultured with a culture
supernatant of 70%(v/v) hybridoma PM-1 and 14000 CPM of
.sup.125I-labeled IL-6 at room temperature for one hour in the
presence of a 100-fold excess of non-labeled IL-6. Seventy
microliters of a sample was layered onto 300 .mu.l of FCS in a 400
.mu.l microfuge polyethylene tube, centrifuged, and then the
radioactivity of the cells was measured. The result revealed that
the antibody produced by the hybridoma PM-1 inhibits the binding of
IL-6 to IL-6 receptor.
Reference Example 2
Construction of a Reshaped Human PM-1 Antibody
[0172] A reshaped human PM-1 antibody was obtained by the method
described in International Patent Application WO 92-19759. From the
hybridoma PM-1 prepared in Reference example 1, total RNA was
prepared in the conventional method, from which single-stranded
cDNA was synthesized. By the polymerase chain reaction (PCR)
method, DNA encoding the V region of mouse PM-1 antibody was
amplified. The primers used in the PCR method are those described
in Jones, S. T. et al., Bio/Technology (1991) 9, 88-89, 1991.
[0173] The PCR-amplified DNA fragments were purified to obtain DNA
fragments containing the gene encoding the V region of mouse
kappa-type L chain and DNA fragments containing the gene encoding
the V region of mouse gamma-type H chain. These DNA fragments were
ligated to a plasmid pUC19, which was then transfected into
competent E. coli cells CH5.alpha. to obtain an E. coli
transformant. From the transformant thus obtained, the above
plasmid was obtained, and the base sequence of the V region-coding
region in the plasmid was determined in a conventional method, and
the complementarity determining region (CDR) of each V region was
identified.
[0174] In order to construct vectors that express chimera PM-1
antibody, cDNAs encoding the V region of .kappa. L chain and H
chain of mouse PM-1 were separately inserted into HCMV expression
vectors. In order to construct a reshaped human PM-1 antibody, the
CDR of V region of mouse PM-1 was implanted to human antibody by
the CDR grafting method. In order for the CDR of human antibody to
form appropriate antigen-binding sites, substitution of amino acids
of the framework region (FR) of antibody V region was
conducted.
[0175] In order to express genes of L chain and H chain of the
reshaped human PM-1 antibody thus constructed, DNA encoding the L
chain or the H chain was separately inserted into a vector
containing the human elongation factor 1.alpha. (HEF-1.alpha.)
promoter, and a vector expressing the L chain or the H chain of the
reshaped human PM-1 (hPM-1) antibody was constructed. By
simultaneously inserting these two expression vectors into CHO
cells, a cell line that produces reshaped human PM-1 antibody
(hPM-1) was established. The ability of hPM-1 thus obtained to bind
to human IL-6 receptor was confirmed by ELISA. Furthermore, hPM-1
inhibited the binding of human IL-6 to human IL-6 receptor in a
similar manner to that of mouse antibody and chimeric antibody.
Example 1
The Effects of Combined Use of Anti-human IL-6 Receptor Antibody
and a Chemotherapeutic Agent on the Growth of Human Myeloma
Cells
[0176] The effects of anti-human IL-6 receptor antibody on
sensitivity of KPMM2 cells to chemotherapeutic agents used for
treatment of myeloma such as adriamycin (ADR, manufactured by Kyowa
Hakko), vincristine (VCR, manufactured by Sigma Chemical Co.), and
melphalan (L-PAM, manufactured by Sigma Chemical Co.) were
evaluated.
[0177] KPMM2 is a multiple myeloma cell line derived from the
ascites of a human patient with myeloma (see Japanese Unexamined
Patent Publication (Kokai) No. 7-236475). The patient with myeloma
had maintained remission by the MCNU (ranimustine) and MP
(melphalan, prednisolone) therapy, but the disease recurred and the
subsequent VAD (vincristine, adriamycin, dexamethasone) therapy was
ineffective. The growth of KPMM2 cells is promoted by IL-6 and is
markedly inhibited by anti-IL-6 antibody or anti-IL-6 receptor
antibody (Rinsho Ketueki [The Japanese Journal of Clinical
Hematology] (1994) 35, 1361-1365). The growth activity of the cell
was evaluated by incorporation of .sup.3H-labeled thymidine
(manufactured by Amersham) into the cell.
[0178] The KPMM2 cells that had been maintained were washed
thoroughly with a fresh medium (RPMI1640 medium supplemented with
20% FBS), and then were adjusted to 4.times.10.sup.5/ml, which was
dispensed in 50 .mu.l aliquots in a 96-well flat-bottomed
microtiter plate (manufactured by Falcon). Furthermore, a medium
containing a recombinant human IL-6 (Asagoe, Y. et al.,
Bio/Technology (1988) 6, 806-809), anti-human IL-6 receptor
antibody hPM-1 (see the above reference example 1 or International
Patent Application WO 92-15759), and the above chemotherapeutic
agents or a fresh medium as control were added to make 200 .mu.l in
each well.
[0179] The plate was incubated at 37.degree. C. for 4 days under
the humidified condition in the presence of 5% CO.sub.2. At four
hours before the end of incubation, 10 .mu.l of .sup.3H-labeled
thymidine solution (100 .mu.Ci/ml) was added to each well, and then
incubated for more 4 hours. At the end of incubation, the cells
were collected onto the glass filter (manufactured by Printed
Filtermat A, WALLAC) using a harvester (Micro 96 Harvester,
manufactured by SKATRON Instruments). The radioactivity
incorporated into the cells was measured by a microbeta (1450
MicroBeta, manufactured by WALLAC).
[0180] The activity of growth inhibition on KPMM2 was expressed
using the effect by a chemotherapeutic agent alone as the control.
Using the incorporation of .sup.3H-labeled thymidine into the cell
for each experimental group in which a each concentration of a
chemotherapeutic agent was added as 100, the amount of
.sup.3H-labeled thymidine incorporated into the cell in an
experimental group in which anti-human IL-6 receptor antibody was
simultaneously added was compared as an index.
[0181] As a result, comparison of the relationship with the
concentration of a chemotherapeutic agent in the presence of a
fixed concentration of anti-human IL-6 receptor antibody revealed
that the index of adriamycin and vincristine was almost constant
regardless of their concentration (FIG. 3 to FIG. 6), whereas the
index decreased with the increase in melphalan concentration (FIG.
1 and FIG. 2). In the presence of 1 ng/ml IL-6, the growth index of
10 .mu.g/ml anti-IL-6 receptor antibody alone was 33.9, whereas it
dropped to 15.5 in the co-existence of 1 .mu.g/ml melphalan.
Similar results were obtained in the presence of 0.1 .mu.g/ml of
IL-6 and the index of the antibody alone was 28.4, whereas in the
co-existence of 1 .mu.g/ml melphalan, it was 15.9. Thus, combined
use of anti-IL-6 receptor antibody and melphalan was shown to have
a synergistic effect.
Example 2
The Effects of Combined Use of Anti-human IL-6 Receptor Antibody
and a Chemotherapeutic Agent in a Human Myeloma Cell-implanted SCID
Mouse System
[0182] It was shown in working example 1 that anti-human IL-6
receptor antibody enhances the antitumor effect of chemotherapeutic
agents. Among them, melphalan (manufactured by Sigma Chemical Co.)
which was found to act in a synergistic manner was used to study an
in vivo effect of combined use.
[0183] For evaluation of antitumor activity, a xenograft model was
used. Thus, KPMM2, a human myeloma cell line derived from ascites
of a patient with multiple myeloma was implanted to a male SCID
mouse (FOX CHASE C. B17/Icr-Scid Jcl, purchased from Nippon Klea)
via the tail vein. At this time, the tumor cells grow in the bone
marrow and come to produce myeloma protein (M protein) in the
peripheral blood. Furthermore, this model system is very close to
an actual clinical condition in that it develops major symptoms of
multiple myeloma in humans such as bone disorders, elevated blood
calcium, and the like.
[0184] Single cell suspension of myeloma cells for implantation was
prepared by passing through a mesh the well minced KPMM2 cells that
have been maintained in vivo. Cell density was adjusted to
3.times.10.sup.7/ml, which was implanted at 0.2 ml per mouse via
the tail vein (6.times.10.sup.7 cells per mouse). The day when the
cells were implanted was set at day 0.
[0185] With regard to anti-human IL-6 receptor antibody, a stock
solution that had been preserved at 12.1 mg/ml was diluted in
sterile phosphate buffer to make 5 mg/ml. This was given to mice at
0.2 ml per animal via the tail vein on day 8 (1 mg per mouse). The
control group received sterile phosphate buffer containing no
antibody in a similar manner.
[0186] Melphalan (L-PAM, manufactured by Sigma Chemical Co.) that
was suspended at 0.3 or 0.1 mg/ml in 0.2% CMC
(carboxymethylcellulose) solution in water was used. This was given
orally at 0.1 ml per 10 g body weight of mouse (3 or 1 mg/kg
weight) for 5 consecutive days starting on day 1. The control group
received 0.2% CMC solution in water containing no melphalan in a
similar manner.
[0187] The experiment was carried out on the following six groups:
(1) the melphalan and antibody non-administration group; (2) the 1
mg/kg melphalan single-drug administration group; (3) the 3 mg/kg
melphalan single-drug administration group; (4) antibody
single-drug administration group; (5) the 1 mg/kg melphalan and
antibody combined administration group; and (6) the 3 mg/kg
melphalan and antibody combined administration group. Group 1
included 9 mice, whereas each of the other groups included 7 mice.
Furthermore, tumor was not implanted on mice of the same lineage
and purchased on the same day, which were used as negative control
for M protein detection.
[0188] Indices of the drug efficacy included the survival period,
the survival ratio without disease on day 120, and the amount of M
protein on day 30. For analysis using a survival curve, a
generalized Wilcoxon's test (SPSS for windows ver. 6, SPSS inc.)
was used. A significance level of 5% or lower was considered to be
significant.
[0189] Serum M protein was detected as human IgG using the ELISA
method. First, mouse serum was dispensed into a 96-well micro plate
that had previously been coated with anti-human IgG antiserum and
was allowed to stand. Then alkaline phosphatase-conjugated human
IgG antibody was bound thereto, and SIGMA104 phosphatase substrate
was added for color development, absorbance of which was read using
a microplate reader. M protein content in the serum was calculated
using the standard curve obtained from normal human IgG.
[0190] In the anti-IL-6 receptor antibody single-drug
administration group or the 1 mg/ml melphalan administration group,
no life elongation effect was observed as compared to the
non-administration group. But the combined use of them
significantly elongated life span as compared to the
non-administration group or both of the single-drug administration
groups (FIG. 7). Furthermore, in the measurement of M protein in
the serum on day 30 after implantation, the combined use of them
reduced M protein level (FIG. 8). In the case of 3 mg/kg melphalan,
a significant life elongation effect was observed for the melphalan
single-drug administration group as compared to the
non-administration group, but by using antibody in combination a
significant life elongation effect was even observed compared to
the single-drug administration (FIG. 9). The survival ratio without
disease on day 120 was 2/7 in the 3 mg/kg melphalan single-drug
administration group, but the ratio improved to 4/7 in the antibody
combined administration group (Table 1).
[0191] The administration of melphalan caused toxicity in mice and
inhibited body weight gain (FIG. 10). When melphalan was used in
combination with anti-IL-6 receptor antibody, an antitumor effect
was enhanced but it did not expand toxicity (inhibition of body
weight gain).
1TABLE 1 Extension of survival period by combined use of anti-human
IL-6 receptor antibody hPM-1 and melphalan Life- None hPM-1
Melphalan Survival elongation disease administration dosage n
period ratio* (%) ratio -- CMC control 9 46.9 .+-. 1.5 100 0/9 -- 1
mg/kg 7 47.3 .+-. 3.1 101 0/7 -- 3 mg/kg 7 84.1 .+-. 9.1 175 2/7
day 8 CMC control 7 55.1 .+-. 5.2 118 0/7 day 8 1 mg/kg 7 68.6 .+-.
7.5 146 0/7 day 8 3 mg/kg 7 110.0 .+-. 5.1 235 4/7 Life elongation
ratio*: 100 .times. (drug administration group/drug
non-administration group). Survival period was expressed as mean
.+-. standard error.
[0192] It was revealed that by using anti-IL-6 receptor antibody
and melphalan in combination, the life elongation effect was
significantly enhanced in KPMM2 which is a cell line derived from a
patient who was resistant to MP and VAD therapy.
Example 3
The Effects of Combined Use of Anti-human IL-6 Receptor Antibody
and a Chemotherapeutic Agent in a Human Myeloma Cell-implanted SCID
Mouse System--a Study on in Vivo Dose Dependency of Antibody
[0193] Example 2 has shown that anti-human IL-6 receptor antibody
enhances the antitumor effect of melphalan. Accordingly, we studied
dose dependency of anti-human IL-6 receptor antibody in the
combined administration of anti-human IL-6 receptor antibody and
melphalan.
[0194] For evaluation of antitumor effects, a xenograft model
animal that was produced by implanting KPMM2 cells via the tail
vein was used as in Working example 2. Thus, single-cell
suspensions which were prepared by passing through a mesh KPMM2
cells that were minced after having been maintained in vivo were
implanted at 0.2 ml per mouse via the tail vein (6.times.10.sup.6
cells per mouse). The day when the cells were implanted was set at
day 0.
[0195] With regard to anti-human IL-6 receptor antibody hPM-1, a
stock solution that had been preserved at 6.57 mg/ml was diluted in
sodium phosphate buffer to make each solution of 5, 1, 0.2, and
0.04 mg/ml. They were given to mice at 0.1 ml per 10 g body weight
of mouse via the tail vein on day 14 to create the 50, 10, 2, and
0.4 mg/kg administration groups. The control group received the
same buffer without antibody in a similar manner.
[0196] Melphalan (L-PAM, manufactured by Sigma Chemical Co.) was
used as a suspension of 0.1 mg/ml in 0.2% CMC solution in water.
This was given orally at 0.1 ml per 10 g body weight of mouse (1
mg/kg weight) for 5 consecutive days starting on day 7. The control
group received 0.2% CMC solution in water without melphalan in a
similar manner.
[0197] The experiment was carried out on the following 10 groups:
(A) the melphalan and the antibody non-administration group; (B)
the melphalan single-drug administration group; (C) the each dose
of antibody single-drug administration group, 4 groups (50, 10, 2,
0.4 mg/kg weight); and, (D) the melphalan and the each dose of
antibody combined administration group, 4 groups (50, 10, 2, 0.4
mg/kg weight). The non-administration group included 12 mice per
group, the melphalan administration group 6 mice per group, whereas
each of the other groups included 7 mice. Furthermore, mice of the
same lineage and purchased on the same day were raised without
implanting a tumor, and the animals were used as negative control
for M protein detection. The amount of M protein was calculated as
described in Example 2.
[0198] Indices of the drug efficacy included the survival period,
and the amount of M proteins on day 30, day 35, and day 42. For
analysis of the survival period, a generalized Wilcoxon's test
(SPSS for Windows ver. 6, SPSS inc.) was used. A significance level
of 5% or lower was considered to be significant. For analysis of
the amount of M protein in the serum, the ANOVA (Analysis of
variance, SPSS for windows ver. 6,. SPSS inc.) was first conducted.
After confirming significance, Bonferroni method (SPSS for windows
ver. 6, SPSS inc.) was used, and a significance level of 5% or
lower was considered to be significant.
[0199] Since death cases occurred on day 35 in the
non-administration group and the each dose of antibody single-drug
administration group, they were compared for the amount of M
protein on day 30. On the other hand, in the melphalan single-drug
administration group, and the melphalan and antibody combined
administration group, the amount of M protein on day 30 was very
low. Since the anti-human IL-6 receptor antibody that had been
administered was detected as M protein thus affecting the assay,
they were compared for the data on day 35 and day 42.
[0200] On day 30 in the antibody single-drug administration group,
none of the doses significantly inhibited the amount of M protein,
but the single-drug administration of melphalan significantly
inhibited this (FIG. 11). Then, on day 35 and 42, the effect of
combined use of antibody administration relative to the melphalan
administration was studied. The result revealed that the combined
use of anti-human IL-6 receptor antibody at 10 mg/kg, 2 mg/kg, and
0.4 mg/kg significantly reduced the amount of M protein (FIG. 12,
13).
[0201] For the survival period, none of the doses in the antibody
single-drug administration group gave a significant life elongation
effect. Furthermore, melphalan alone has shown a significant life
elongation effect, which was further enhanced by combined use of
antibody (Table 2). At any dose, the life elongation effect tended
to increase. Furthermore, in the 0.4 mg/kg and 50 mg/kg
administration groups, significance was observed relative to the
melphalan single-drug administration group in the generalized
Wilcoxon's test (FIG. 14).
2TABLE 2 Extension of survival period by combined use of anti-human
IL-6 receptor antibody hPM-1 and melphalan Life- hPM-1 Melphalan
Survival elongation administration dosage n period ratio (%) -- CMC
control 12 41.0 .+-. 1.7 100 50 mg/kg CMC control 7 41.6 .+-. 1.0
101 10 mg/kg CMC control 7 40.4 .+-. 1.7 99 2 mg/kg CMC control 7
41.4 .+-. 1.3 101 0.4 mg/kg CMC control 7 40.3 .+-. 1.2 98 -- 1
mg/kg 6 58.2 .+-. 1.8 142 (100) 50 mg/kg 1 mg/kg 7 65.7 .+-. 3.9
160 (113) 10 mg/kg 1 mg/kg 7 64.3 .+-. 2.7 157 (111) 2 mg/kg 1
mg/kg 7 63.3 .+-. 2.3 154 (109) 0.4 mg/kg 1 mg/kg 7 63.7 .+-. 1.1
155 (110) Life elongation ratio: 100 .times. (drug administration
group/drug non-administration group). Values in the parentheses
were obtained using the melphalan single-drug administration group
as control. Survival period was expressed as mean .+-. standard
error.
[0202] From the foregoing, it was demonstrated, the combined use of
anti-IL-6 receptor antibody and melphalan has shown an antitumor
effect at any dose from 0.4 mg/kg through 50 mg/kg.
[0203] The administration of melphalan caused toxicity to mice and
inhibited body weight gain. When melphalan was used in combination
with anti-human IL-6 receptor antibody, an antitumor effect was
enhanced but toxicity (inhibition of body weight gain) was not
expanded. Therefore, it was suggested that in the treatment of
myeloma the administration of melphalan may be useful in enhancing
effects, reducing the dosage, and breaking
melphalan-resistance.
[0204] Reference to microorganisms deposited under Patent
Cooperation TReaty Rule 13(2):
[0205] The name and adddress of the depository institute Depository
organization: the National Institute of Bioscience and Human
Technology,
[0206] Agency of Industrial Science and Technology Address: 1-3,
Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan
[0207] Accession No. FERM BP-2998
[0208] Date deposited Jul. 10, 1990
[0209] Depository organization: National Collections of Industrial
and Marine Bacteria Limited
[0210] Address: 23 St Macher Drive, Aberdeen AB2 IRY, UNITED
KINGDOM
[0211] NCIMB 40366 Feb. 11, 1991
[0212] NCIMB 40362 Feb. 11, 1991
* * * * *